The binding of NAD ion, NADH and ADP-ribose to horse live alcohol dehydrogenase (LADH) has been studied calorimetrically as a function of pH at 25 degrees. The enthalpy of NAD ion binding varies with pH in a sigmoidal fashion and is -4.0 kcal mol (NAD) -1 at pH 6.0 and +4.5 kcal mol (NAD) -1 at pH 8.6 with an apparent pk of 7.6 plus or minus 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8-9.8 kcal mol (H+)-1. In conjunction with the available thermodynamic data on the ionization of Zn-bound water in model compounds it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme-NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with the zinc-free enzyme provide further evidence for this conclusion.